The amount of calcium deposited in the matrix of the substitutes after 35 days of culture was quantified using an o-cresolphthalein complexone method [43 (link)]. Briefly, 5 mm diameter punch biopsies of the substitutes were collected for each group. Samples were decalcified in 0.6 N HCl (1 mL) for 48 h at 4 °C. After brief mixing and centrifugation, each extract supernatant (100 μL) was mixed with an ammonium chloride buffer (2.8% NH4OH, 24.9 × 10−3 M NH4Cl, pH 10.5), 8-quinolinol (0.22%), and o-cresolphthalein complexone solution (0.022%) (Sigma-Aldrich). The level of calcium in each sample was determined from a standard curve of known calcium concentrations measured at 575 nm (SPECTRA Plus 384, Molecular Devices). Samples were run in duplicate for each condition (n = 3 tissues/condition), and data were normalized to the punch biopsy area.
Ammonium chloride buffer
Manufactured by Merck Group
Sourced in United Kingdom
Ammonium chloride buffer is a laboratory reagent used to maintain a specific pH level in aqueous solutions. It is a mixture of ammonium chloride (NH4Cl) and ammonia (NH3) that acts as a pH buffer, helping to stabilize the pH of a solution within a certain range. The core function of this buffer is to provide a stable, controlled pH environment for various chemical and biological applications.
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Lab products found in correlation
8 quinolinol, by Merck Group (1 mentions)
Anti cd86 apc, by BD (1 mentions)
Anti cd4 apc, by BD (1 mentions)
Anti cd11b af700, by BD (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Anti cd4 af700, by BD (1 mentions)
Anti cd62l percp cy5, by BD (1 mentions)
Anti cd44 apc cy7, by BD (1 mentions)
Facs buffer, by Merck Group (1 mentions)
Anti cd80 pe, by BD (1 mentions)
Anti cd11c pe cy7, by BD (1 mentions)
Anti cd45.1 fitc, by BD (1 mentions)
Sodium azide, by Merck Group (1 mentions)
Anti ki67 pecy7, by BD (1 mentions)
Z2 coulter counter, by Beckman Coulter (1 mentions)
Dnase 1, by Roche (1 mentions)
Collagenase d, by Roche (1 mentions)
Liberase tl, by Roche (1 mentions)
3 protocols using ammonium chloride buffer
1
Quantifying Calcium Deposition in Tissue Substitutes
2
Multi-parameter Flow Cytometry Analysis
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Single cell suspensions were made from the spleen and draining lymph nodes, red blood cells were lysed using an ammonium chloride buffer (Sigma Aldrich, Dorset, UK), and cells were then re-suspended in FACS buffer (PBS, 2% fetal calf serum, 0.01% sodium azide (Sigma Aldrich, Dorset, UK). Fc receptors were blocked with supernatant from the hybridoma 2.4G2. All antibodies were from eBioscience, Hatfield, UK, except where stated; live/dead fixable cell stain conjugated to ef455 (Life Technologies), anti-CD4-APC, anti-CD4-AF700 (BD Pharmingen, Oxford, UK), anti-CD11c-PE-Cy7, anti-CD11c-ef450, anti-Ki67-PE-Cy7, anti-CD11b-Af700, anti-CD45.1-FITC, anti-CD44-APC-Cy7, anti-CD80-PE, anti-CD86-APC, anti-CD62L-PerCP-Cy5.5, and anti-Foxp3-ef450. FACS data were collected using a 6 laser LSR Fortessa (BD Biosciences, New Jersey, USA) and analyzed using FlowJo software (Tree Star, Olten, Switzerland).
3
Bronchoalveolar Lavage and Lung Tissue Dissociation
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Mice were sacrificed by cervical dislocation. The trachea was exposed and cannulated with a 21-gauge needle encased in a silicon tube. Lungs were lavaged three times with 1 mL of cold PBS. Samples were centrifuged at 300 × g for 5 minutes at 4°C, and supernatant from the first lavage was stored in −80°C for cytokine detection. Cell counts were performed by using a Z2 Coulter counter (Beckman Coulter, Fullerton, CA), according to manufacturer's instructions. Single-cell suspensions were used for further analysis.
After performing BAL, lungs were perfused via the right ventricle of the heart with 5 mL PBS to deplete the intravascular pool of cells from the lung vasculature. Lung and lung dMLNs were removed, cut into pieces, and digested in RPMI 1640 medium containing DNase I (0.2 mg/mL) and Liberase TL (0.33 mg/mL) or collagenase D (2 mg/mL) (all from Roche, Welwyn Garden City, UK) for 30 minutes at 37°C and then incubated in PBS/0.5% bovine serum albumin/10 mmol/L EDTA for another 5 minutes. Single-cell suspensions were prepared from the predigested tissues by passing through 40-μm cells strainers (BD Falcon, Devon, UK) and washed cell strainer with PBS/0.5% bovine serum albumin/2 mmol/L EDTA. Red blood cells were lysed by using ammonium chloride buffer (Sigma). Cell count was performed with a Z2 Coulter counter.
After performing BAL, lungs were perfused via the right ventricle of the heart with 5 mL PBS to deplete the intravascular pool of cells from the lung vasculature. Lung and lung dMLNs were removed, cut into pieces, and digested in RPMI 1640 medium containing DNase I (0.2 mg/mL) and Liberase TL (0.33 mg/mL) or collagenase D (2 mg/mL) (all from Roche, Welwyn Garden City, UK) for 30 minutes at 37°C and then incubated in PBS/0.5% bovine serum albumin/10 mmol/L EDTA for another 5 minutes. Single-cell suspensions were prepared from the predigested tissues by passing through 40-μm cells strainers (BD Falcon, Devon, UK) and washed cell strainer with PBS/0.5% bovine serum albumin/2 mmol/L EDTA. Red blood cells were lysed by using ammonium chloride buffer (Sigma). Cell count was performed with a Z2 Coulter counter.
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